Combining Nanoparticle Shape Modulation and Polymersome Technology in Drug Delivery

Abstract

This paper highlights the potential benefits of using self-assembled polymeric nanoparticles of various shapes to enhance drug uptake. First, we highlight the growth and development of the polymersome, using a liposome as a blueprint for amphiphilic codelivery. Then, we focus on the advantages of nanoparticle elongation, drawing from the field of solid nanoparticles, as opposed to self-assembled vesicles which have not yet been extensively explored in shape-modulated drug delivery applications. Notably, regardless of the material used in the solid nanoparticle systems, more elongated shapes lead to greater cellular uptake, decreased interaction with the reticuloendothelial system macrophages, and increased circulation times. Finally, we highlight the methods currently being developed to modulate polymersome shape, thus providing a drug delivery system with the benefits derived from amphiphilicity and elongated structures. Current methods employed to modulate polymersome shape involve osmotic pressure gradients, solvent switching, and the use of cross-linking agents. Although these methods are successful in modulating polymersome shapes and the benefits of elongated nanoparticles in therapeutic targeting are clear, these methods have not yet been explored for applications in drug delivery.

Graphical Abstract

1. INTRODUCTION

Nanotechnology, though still an emerging science, has been attracting attention on many fronts since its initial discovery in the 1980s.¹ One of the most notable applications, and the focus of this review, is its potential to advance targeted drug delivery in biological systems. At the forefront of this application are self-assembled nanoparticles, which have natural equilibrium structures, based on component hydrophobicities,² and can encapsulate and protect long-term circulating drugs.³ Self-assembled nanoparticles that have proven most useful thus far in drug delivery are micelles, liposomes, and polymersomes.^4,5 Each of these thermodynamically self-assembled nanoparticles have unique structural profiles that contribute to their respective successes and shortcomings in attaining the perfect cocktail of bioavailability, enhanced circulation time, molecular targeting, controlled release, and protection of the drug they carry.⁶

Although self-assembled particles have already shown great promise due to ease in formation and well understood assembling behavior,^7,8 regardless of the type, the most commonly utilized shape of a sphere is not always ideal for cellular internalization. Notably, elongated structures, as opposed to spherical, more adequately mimic native biologic targets, leading to enhanced uptake^9–11 in areas of margination experienced in larger diameter blood vessels.¹² Despite clear advantages to the use of self-assembled systems and an understanding of what drives self-assembly of micelles into elongated shapes,¹³ it is not yet an established science as to how to alter bilayer systems like polymersomes into elongated equilibriums. Drawing from solid nanoparticles, whose success in modulating shape to enhance drug delivery will be documented in this review paper, self-assembled systems are being altered in shape to incorporate the additional benefits of amphiphilicity to the established benefits of changing shape.

Micelles and liposomes are among the most common nanoparticles explored preclinically. In addition to their high potential as drug delivery mechanisms, there is evidence that with minor changes in design, the efficacy of these particles in terms of circulation, targeting, uptake, and toxicity can be increased significantly. Additionally, the manipulation of these two particles over the years has acted as a blueprint for the formation, modulation, and further investigation of the polymersome.

Like liposomes and micelles, the polymersome has both hydrophobic and hydrophilic tendencies due to its amphiphilic make up that make it a universal vessel in therapeutic drug delivery. In comparison to micelles and liposomes, polymersomes possess increased long-term stability and decreased loss of valuable encapsulated drug.^14–16 This multifaceted set of amphiphilic advantages produces a unique architecture that allows both the encapsulation and release of hydrophobic drugs while also maintaining a hydrophilic core and brush to minimize protein absorption and protect the vessel from biological invasion.¹⁷ While the polymersome does hold a certain number of physiological advantages over its counterparts, it is important to note that they are not perfect. Polymersomes have a notably low lateral diffusivity and are more thermodynamically stable than both micelles and liposomes. This makes the polymeric vessel more susceptible to an immune response, as it less adequately mimics biologic components.¹⁸ The immune system is a major barrier in self-assembled drug delivery across the board; however, manipulation of polymer chemistry, specifically shape modulation, has shown great promise in minimizing immune system contact and maximizing carrying capacity, stability, and targeting capabilities.¹⁸ Similar to the micelles and liposomes, the potency of the polymersome has shown significant growth with a change in geometry. Though the techniques of shape change vary, ranging from dialysis to solvent exchange (discussed in the following sections), the common goal of polymersome shape modulation is to stray away from the conventional sphere and move toward the worm-like or prolate shapes. These shapes have proven more effective in cellular uptake than their cubic and spherical counterparts partly due to the thermodynamic advantages associated with their smaller size and increased surface area.¹⁹ Depending on their target, the particles can be specified even further by changing the protein coating, charge, and geometry to ensure efficacy.¹⁹ It is widely believed that by further modifying these polymersomes to fit those parameters described above with altered shape, in accordance with the needs of the biological system, that the use of self-assembled nanotechnology in drug delivery can offer effective, noninvasive therapeutic advantages to a number of ailments, including cancers, stomach, and GI infections, as well as the potential to cross the blood brain barrier,²⁰ in the future.

2. POLYMERSOMES USED IN DRUG DELIVERY

Using liposomal success as a model for development, polymersomes emerged as alternative systems for vesicular-based delivery of drug products. Following the liposomal blueprint, the polymersome does have a bilayer; however, instead of a phospholipid lining, it utilizes a double layer of synthetic polymers as seen in Figure 1, which creates an added advantage and thermodynamic favorability.^21–24 These vessels have demonstrated preclinical promise due to their modular nature based on polymer selection. Polymer selection can lead to a large solubilization power,²⁵ loading capacity, stability in the bloodstream, therapeutic potential, and longevity.^24,26 Drug delivery via polymersomes could allow for a less toxic and more specific means of getting medication to targeted cells with the use of targeting ligands and bilayer protection without compromising efficiency. These traits, however, would not be as noteworthy without the polymersomes’ diverse potential to be altered in shape after formation. Unlike their counterparts, polymersomes can be synthesized with a wider variety of polymeric materials, with amphiphilic properties, like PEG, pH-responsive polyesters, biologic polymers, and other stimuli-responsive polymers,^15,16,27 with specific targets in terms of shape, size, and sustainability. In preclinical trials by Wei et al. the controlled release of multiple drugs was compared, and it was concluded that polymersomes were able to load both paclitaxel and doxorubicin in tandem and release them in a pH-specific manner.²⁸ Work done by Ghoroghchian et al. shows a similar situation in which the drug doxorubicin is successfully loaded into a polymersome and targeted to release in an acidic environment rather than physiological pH to mimic carcinogenic conditions.²⁸ There has also been documentation of a targeted release based on other stimuli including but not limited to hypoxia, chemical species, and temperature to further personalize the polymersomes’ drug delivery capabilities. With so many combinations to consider, it is important to take note of the synthesis of polymersomes and how this delicate process can be modified to change carrying capacity, target, circulation, release, and toxicity. Understanding the impact of shape change in solid nanoparticles on the ultimate in vivo fate motivates the development of techniques to modify the polymersome shape, which allows for all of the benefits of polymersomes (stimuli-responsive, decreased leakiness, and dual delivery) to be harnessed in tandem with changing shape.

Figure 1.

Comparative properties of liposomes and polymersomes. Liposomes and polymersomes demonstrate similar encapsulation (a). Most notable differences between liposomes and polymersomes occur in stability, noted by liposomal fluidity (c), which leads to increased leakiness (b) and lower stability (d) in comparison to polymersomes.¹⁸ (e) demonstrates the chemical versatility in liposome and polymersome nanoparticles, based on available amphiphiles. Reproduced from ref 18. This article is licensed under a Creative Commons Attribution 3.0 Unported License. Copyright 2018 The Royal Society of Chemistry.

3. BENEFITS OF SHAPE CHANGE IN DRUG UPTAKE

3.1. Shape Change in Solid Nanoparticles.

Most nanoparticles have a spherical shape. With advanced nanofabrication techniques, different shapes of nanoparticles have emerged in recent years with unique geometrical, physical, and chemical properties. For example, nanorods with elongated aspect ratios have been fabricated as a novel contrast agent for both molecular imaging and photothermal cancer therapy;^29,30 asymmetrically functionalized gold nanoparticles have been assembled to build nanochains;³¹ superparamagnetic iron-oxide-based nanoworms are studied for tumor targeting,³² and nanonecklaces are assembled by covalent bonding using gold nanoparticles.³³ It has been reported that cylindrically shaped filamentous micelles can effectively evade nonspecific uptake by the reticuloendothelial system (RES), allowing persistent circulation for up to 1 week after intravenous injection.³³ It is known that spherical particles bigger than 200 nm are efficiently filtered by the liver, spleen, and bone marrow, while particles smaller than 6 nm³⁴ can be quickly cleared by the kidney or through extravasation, thus making 10–200 nm the ideal size range for the circulating spherical carriers.^35–37 However, the role of altering the shape in increasing targeted drug delivery has more slowly emerged over the last 10 to 15 years.

A general trend observed is that more elongated structures have higher residence times and better target endothelium due to margination, regardless of the material makeup of the nanoparticle, although surface coatings can play a role.^38–40 By stretching nanoparticles into a more elongated shape, the surface area is increased, which will provide more surface available for contact with the target, as seen in Figure 2.^29,41

Figure 2.

Adhesion probability of nanoparticles of various aspect ratios based on particle volume. It is important to note that at all particle volumes, spherical nanoparticles have the lowest probability of cellular adhesion, despite their widespread use. Created with BioRender.com. Adapted from refs 29 and 41.

This trend is observed when comparing the uptake of gold nanorods, which are more elongated, to more rounded gold “nanostars”,⁴² where gold nanorods have higher uptake. In another application, mesoporous silica nanoparticles of aspect ratios of 1 (spherical), 1.75 (short rods), and 5 (long rods) were delivered orally, with the goal of intestinal absorption. It was observed that after 7 days, spherical particles were mostly found in excretory organs, namely the liver, and both short-and long-rod nanoparticles were observed in the intestines, indicating increased intestinal residence time and enhanced uptake via oral administration in comparison to spherical counterparts.⁴³ When incubated with Caco-2 intestinal cells, polystyrene rods and discs led to 2-fold increases in uptake in comparison to polystyrene spheres without the addition of conjugation ligands. Upon conjugation, polystyrene rods were taken up at approximately 4 times the mass compared to conjugated polystyrene spheres.⁴⁴ Together, this draws the conclusion that regardless of material, elongation can have positive effects on increasing targeted nanoparticle uptake. There is, however, a limit to the benefit of elongating nanoparticles when using intravenous injection, as elongated filomicelles with aspect ratios greater than 20 can be carried further via blood flow instead of internalized and accumulated in diseased areas in vivo.⁴⁵

More elongated structures also have the potential to evade the immune system, with the local shape at the point of contact between the nanoparticle and macrophages dictating engulfment or spreading of polystyrene particles.⁴⁶ If polystyrene rods came into contact with macrophages on their elongated edge, they were less likely to be engulfed (Figure 3), noting the shape-driven in vitro effect. Similar behavior is observed with elongated silica nanoparticles, showing decreased uptake by the reticuloendothelial system compared to their spherical counterparts.⁴⁷ However, polystyrene rods still do illicit an immune response when incubated with DC2.4 dendritic cells, although it is less prominent than the immune response caused by spherical particles around 193 nm in diameter.²⁸ A recent study by Visalakshan et al. suggests that this immune evasion may be due to enhanced adsorption of beneficial proteins, immunoglobulins, forming a more dense protein corona around elongated particles, compared to a smaller quantity of protein adsoprtion observed on spherical particles.⁴⁸ Simultaneously, elongated particles presented with decreased adsorption of albumin compared to spherical particles, emphasizing the effect that shape has on protein adsorption.⁴⁸ Elongation may not be the only important factor with regards to evading the immune system. A study by Devarajan et al. observed that “irregular”-shaped polymer lipid nanoparticles preferentially accumulated into the spleen instead of being excreted via the liver, thus evading the standard immune clearance of Kupfer cells.²⁸

Figure 3.

Engulfment of polystyrene nanoparticles as observed by scanning electron microscopy (A–C). Most noteworthy is the observed spreading of the macrophage (brown) over the polystyrene rod (purple) when the interaction occurs on the elongated side of the particle (B). Figure adapted from Champion et al.⁴⁶ Reproduced with permission from ref 46. Copyright 2006 National Academy of Sciences, U.S.A.

The idea of utilizing different nanoparticle shapes for enhanced biologic uptake in a specific diseased area is not a new one. Kolhar et al. found that polystyrene nanorods showed higher targeted uptake toward the endothelium compared with spherical particles, confirmed by both in vitro and in vivo assays.⁴⁹ Furthermore, the greatest discrepancy in uptake was observed in the brains of mice, where targeted polystyrene rods, labeled with a molecular antibody to intracellular adhesion molecule (ICAM), were taken up in 7.5× greater amounts than targeted polystyrene spheres.⁴⁹ Biologically, elongated structures seem to have a more promising internal fate in the nucleus, likely based on an increased interaction with receptors leading to increased cellular internalization. In one study, rods and worms made from polymeric material poly(oligoethylene glycol methacrylate)-block-poly(styrene-covinyl benzaldehyde) were shown to passively diffuse through the cellular membrane of MCF7 human breast cancer cells and deliver doxorubicin to the nucleus, while more spherical shapes were less effective.⁴⁰

All of this together indicates that elongated structures are desirable and can be designed for more site-specific delivery, in part due to evasion of typical immune system responses and enhanced endothelial uptake due to the margination effect experienced. Extending this shape effect to self-assembled, bilayer polymeric systems, polymersomes, will provide the benefits of both and has the potential to reach currently untreatable areas of the body.

3.2. Shape Change in Polymersomes.

3.2.1. Methods of Polymersome Shape Change.

Self-assembled systems explored in drug delivery tend toward spherical shapes, as this bilayer can lead to protection of valuable payloads. Spherical polymersomes can become kinetically trapped. Therefore, forming alternative shapes from self-assembled polymersomes requires the addition of one or multiple forces to shift the system out of the thermodynamically stable or kinetically trapped spherical shape. These additional pressures need to overcome bending energy in order to make vesicles change shape and elongate. Bending energy is determined by eq 1, in which k (bending rigidity) and C₀ (spontaneous curvature) are expected to change based on polymer and microenvironment changes. C (mean surface curvature) is dependent on the final shape.²⁵

$$E_{\text{b}}=\frac{k}{2}{\displaystyle \oint {\left(2C-C_0\right)}^2\text{d}A}$$ (1)

The minimum bending energy to initiate a shape change clearly relates to the pressure within the vesicle, which can be controlled by osmotic pressure gradients in formation solution.⁵⁰

There are many methods to alter the shape of polymersomes to become more elongated by overcoming this bending energy. Most methods of altering the polymersome shape can fall into two major categories: (1) a solvent-driven osmotic pressure change or (2) a salt-driven osmotic pressure change. Both methods see a decrease in the internal vesicle volume as water flows out to balance the osmotic pressure gradient introduced by the addition of either solvent or salt in the solution outside of the polymersome. A solvent-driven shape change can lead to more long-term stability, by kinetically trapping the polymersome through hydrophobic membrane plasticizing. Table 1 summarizes polymersome shape change presented in the literature thus far, organized by the final shape obtained. In the sections below, we highlight some of the major methods of polymersome shape change.

Table 1.

Summary of Shape-Modulated Polymersomes, with a Focus on Elongated Systems That Have the Potential to Increase Targeted Drug Delivery Applications

	shape	materials	method of preparation	citation
elongated	ellipsoids	polyethylene glycol-b-poly(N-isoproylacrylamide-co-perylene diester monoimide)	THF solvent and perylene group directed	59
		polyethylene glycol-b-polystyrene	dialysis against 10 mM NaSCN, 10 or 100 mM NaNO3, 100 mM NaCl, 10 000 mM CaCl2	57
		polyethylene glycol-b-poly(N-isoproylacrylamide-co-perylene diester monoimide)	solvent switching (THF and water)	63
		poly(2-hydroxyethyl aspartamide)-b-polyethylene glycol	degree of substitution of PEG at 1 mol %	64
	prolates	polyethylene glycol-b-polystyrene	solvent dissolution in 1:1 organic solvent and water	52
		polyethylene glycol-b-polylactic acid	dialysis against 50 mM NaCl	20
		poly(dimethylsiloxane)-g-poly(ethylene oxide)	hypertonic stress with 250 mM glucose	56
	rods	polyethylene glycol-b-polystyrene	solvent induced 1:1 THF to water	54
		polyethylene glycol-b-polystyrene	low amounts of PEG addition combined with solvent dissolution at 10:3 organic/water	65
	tubes	polyethylene glycol-b-poly(N-isoproylacrylamide-co-perylene diester monoimide)	THF solvent (high concentration) and perylene group directed	59
		polyethylene glycol-b-poly(styrene-co-4-vinyl benzyl azide)	cross-linking induced cycloaddition with azide concentrations from ~18 to 23%	61
		polyethylene glycol-b-poly(styrene-stat-(coumarin methacrylate))-b-polyethylene glycol	UV light induced cross-linking	66
		polyethylene glycol-b-polystyrene	dialysis against 1000 mM Na2HPO4, 1000 mM Na2SO4, 1000 mM CaCl2	57
		polyethylene glycol-b-polylactic acid	dialysis against 5 to 50 mM NaCl	25
cell-like	stomatocytes	polyethylene glycol-b-polylactic acid	dialysis against 50 and 100 mM NaCl	53
		polyethylene glycol-b-polystyrene	plasticizing via organic solvent (THF/dioxane 1:1)	51
		polyethylene glycol-b-polybutadiene with poly(acrylic acid)-b-polybutadiene	coassembly of anionic and neutral polymers to increase repulsive forces and, therefore, curvature	58
		polyethylene glycol-b-polystyrene	plasticizing via organic solvent (THF/dioxane) at a 1:1 organic solvent/water ratio	55
		polyethylene glycol-b-polystyrene	dialysis against 10 mM NaCl, 100 mM Na2HPO4, 100 mM Na2SO4, 10 mM NH4Cl, 1000 mM MgCl2, 1000 mM CaCl2	57
		polyethylene glycol-b-polystyrene	high amounts of PEG addition combined with solvent dissolution at 10:3 organic solvent/water	65
	discs and stomatocytes	polyethylene glycol-b-polystyrene	solvent dissolution 3:1 organic solvent and water	52
		polyethylene glycol-b-polylactic acid	dialysis against 10 mM NaCl	53
	discs	polyethylene glycol-b-polystyrene	solvent induced 1:2 THF to water	54
unique	dendrite-like	poly(ethylene oxide)-b-polystyrene	blending homopolymer with copolymer	67
	hexagonally packed hollow hoops	polyethylene glycol-b-polystyrene	10 and 25 mM NaCl salt concentration and azide group directed	68
	polyhedrons	polyethylene glycol-b-poly(N-isoproylacrylamide-co-perylene diester monoimide)	perlyene group direction and 65 to 70% THF	60
	large compound vesicles	polyethylene glycol-b-polystyrene	dialysis against 10 mM Na2HPO4, 10 mM Na2SO4, 10 mM MgCl2, 10 mM CaCl2	57

3.2.1.1. Solvent-Driven Polymersome Shape Change.

One set of methods utilizes the introduction of various solvents to increase osmotic pressure in solution to “bend” the polymersome into the shape desired. One procedure frequently used by many laboratories is the method established by Kim and van Hest,⁵¹ where polymersomes are prepared through standard methods and then plasticized in an organic solvent and water mixture. Organic solvent becomes trapped in the membrane over time and creates an increasing barrier to the transport of water in and out of the polymersome. This solvent addition decreases and then maintains a decreased internal vesicle volume, leading to elongation of the polymersome. Following this, polymersomes are dialyzed against pure water to encourage and solidify shape change through the creation of a new osmotic pressure gradient.

Kim et al. used a 1:1 mixture of tetrahydrofuran (THF)/dioxane and water. After the introduction of solvents, the PEG-b-polystyrene (PS) polymersomes are then extensively dialyzed against water. The organic solvent fluidizes the membrane, allowing for shape change to occur. The rapid diffusion of water across the bilayer membrane and into the surrounding system causes the hydrophobic portion to lose fluidity and recover the glassy state.⁵¹ Depending on the organic solvent to water ratio, different shapes are induced. Solvent ratios were further explored by Rikken et al. (Figure 4) The data collected indicates a 1:3 water to organic solvent ratio creates spheres, a 1:1 water to organic solvent creates prolates, and a 3:1 water to organic solvent ratio creates stomatocytes, confirmed via dynamic light scattering (DLS) and cryo-transmission electron microscopy (TEM).⁵² Pijpers et al. delved deeper into the thermodynamic reasoning behind polymersome elongation, discovering that solvents lead to various PEG chain expansion volumes due to differences in solvation. This change in hydrodynamic chain volume on the external surface of the polymersome can drive changes in C₀ leading to shape change⁵³ (Figure 5). Solvent-based approaches appear to be the most common method for shape modulation of PEG-b-PS polymersomes,^51,52,54,55 while polyester-based polymersomes are more often modulated through salt-driven osmotic pressure gradients.

Figure 4.

Shape change of polymersomes using solvents. (a) Key. (b) Introduction of polymeric amphiphiles in organic solution is followed by (c) the addition of some water, which causes polymersomes to form. Finally, in (d), water is added in excess, trapping the solvent inside of the polymersome and leading to various shapes, depending on conditions, with results represented in (e).⁵² Reproduced from reference 52. This work is licensed under a Creative Commons Attribution 4.0 International License. Copyright 2016 The Authors.

Figure 5.

Effect of (A) spontaneous curvature and (B) PEG surface confirmation on PEG-b-PDLLA polymersome shape modulation.⁵³ Reproduced from reference 53. Copyright 2017 American Chemical Society.

3.2.1.2. Salt-Driven Polymersome Shape Change.

An alternative approach to shape modulation of polymersomes involves the use of salts to create an osmotic pressure gradient as opposed to the solvent-based approach described above. In 2013, Salva et al. identified the role of osmotic pressure to modulate polymersome diameters, using glucose to induce osmotic pressure on poly(dimethyl siloxane)-g-poly(ethylene oxide) (PDMS-g-PEO) polymersomes, noting that hypertonic stress led to vesicle shrinking, while hypotonic stress had the opposite effect and caused the polymersomes to swell. As discussed above, shape modulation is driven by this decrease in internal polymersome volume, which was observed to occur at glucose gradients of 100 and 250 mM, with higher concentrations of glucose leading to further elongation.⁵⁶ L’Amoreaux et al. created polymersomes of polyethylene glycol-b-polylactic acid (PEG-b-PLA) via nanoprecipitation. Following nanoprecipitation, polymersomes are dialyzed overnight to remove organic solvent trapped in the inner bilayer of the polymersome to ensure solidification of the membrane. After the polymersomes are cleared of solvent, they are transferred to a 50 mM NaCl solution, where they are dialyzed overnight to introduce an osmotic pressure gradient and encourage a shape change. DLS suggests a shape change occurred, shown by increases in the polydispersity index (PDI), which is confirmed through TEM.²⁰ Abdelmohsen et al. observed similar behavior when dialyzing PEG-b-PLA polymersomes against salt, seeing increased elongation with increasing NaCl concentrations from 5 to 50 mM.²⁵

Recently, Men et al. explored the role of multiple cations and anions on the shape transformation of formed PEG-b-PS polymersomes. Salts were added rapidly, and in general, it was observed that larger salt concentrations led to bigger osmotic pressure gradients and therefore increased deflation of the polymersomes. Notably, different explored ions led to different shape modulation effects, which was suggested to be due to differences in ion and PEG interactions. PEG-b-PS polymersomes were changed into ellipsoids, stomatocytes, and tubules by different cations (NH₄Cl, NaCl, MgCl₂, and CaCl₂) and anions (NaSCN, NaNO₃, NaCl, Na₂HPO₄, and Na₂SO₄), with results summarized in Table 1. The most interesting conclusion from this research was that the Hofmeister series, a classification of ions in order of their ability to salt in or salt out proteins, could be used to predict shape modulation of polymersomes⁵⁷

3.2.1.3. Other Methods of Polymersome Shape Change.

Although success has been observed and design rules have begun to be established when using solvent- and salt-driven osmotic pressure gradients to modulate the shape of polymersomes, alternative approaches have been explored to increase control over the shape change process. One chargebased approach uses coassembly of two block copolymers, poly(acrylic acid)-b-polybutadiene (PAA-b-PBD) as an anionic polymer and PEG-b-PBD as a neutral polymer. The addition of the anionic PAA chain at a 25 to 75 molar ratio of PAA-b-PBD to PEG-b-PBD allowed for an increase in repulsive forces and therefore an increase in curvature compared to PEG-b-PBD-alone polymersomes, leading to the formation of stomatocytes. Upon the addition of positively charged calcium ions, 25:75 PAA-b-PBD/PEG-b-PBD stomatocyte polymersomes changed to a more spherical shape, indicating the importance of the anionic PAA in increasing the polymersome curvature.⁵⁸ Che et al. used a similar approach by introducing azide groups to the chain ends of PEG in PEG-b-PS polymersomes in tandem with salt-driven osmotic pressure approaches, where the contribution from azide increases the internal hydrodynamic PEG surface volume in comparison to the external PEG surface volume and causes an internal collapse and the formation of internal structures, identified by the authors as hexagonally packed hollow hoops.⁵⁸

A similar observation was noted by Wong et al. in 2017, where by the introduction of perylene rings in polymersomes, with a block copolymer basis of PEG-b-poly(N-isopropylacrylamide-co-perlyene diester monomide) (PEG-b-P(NIPAM-co-PDMI)), the polymersome shape could be fine-tuned through changing the solvent concentration. This approach combines solvent-driven shape modulation with the addition of chemical alternations to the polymersome surface to enhance aggregation. As noted in Figure 6, elongation was controlled by concentrations of THF.⁵⁹ Exploring this further, Wong et al. increased the amount of PDMI in PEG-b-P(NIPAM-co-PDMI, where PDMI introduces perylene aggregation when in the presence of THF. After forming polymersomes, PEG-b-P(NIPAM-co-PDMI) shifted into polyhedrons in the presence of 65 and 70% THF in water, which the authors attribute to the plasticizing nature of THF in combination with perylene aggregation.⁶⁰

Figure 6.

TEM images of PEG-b-P(NIPAM-co-PDMI) via increasing THF concentration post formation indicates control over the shape modulation process.⁵⁹ Reproduced from reference 59. This article is licensed under a Creative Commons Attribution 4.0 International License. Copyright 2017 The Authors.

Another strategy used to induce shape change is the use of chemical addition. The method utilizes covalent cross-linkers. Not only are polymersomes sensitive to osmotic gradients but also they can change their shape in response to chemical stimuli. Although it is useful, the method can be difficult to reproduce, associated with the rigor required to build the copolymer backbone. The van Hest group incorporated azide handlers and bicyclo[6.1.0]nonyne (BCN) into the block copolymer of PEG-b-P(styrene-co-4-vinyl benzyl azide), which upon mixing can cross-link with one another via a strained-promoted alkyne–azide cycloaddition (SPAAC) reaction and leads to tubular polymersomes.⁶¹ More methods for creating polymersomes are summarized in Table 1. Notably, Table 1 also summarizes the formation of unique polymersome shapes that are beyond the major scope of this paper.

3.2.2. Benefits of Shape-Changed Polymersomes in Drug Delivery Applications.

As discussed above, polymersomes change shape when their membranes are kinetically manipulated to retain certain shapes at a specific time during synthesis.⁵¹ The transition from the sphere to the kinetically favored nanorod, stomatocyte, or nanonecklace, occurs under the influence of specific solvents or salts.¹⁴ Each unique morphology comes with its own advantages and disadvantages, but for this paper, we focus on the profiled more elongated nanorods or prolates, for the aforementioned reasons above. Specifically, the prolate shape has shown great promise in longevity, decreased toxicity, targeting specificity, and loading capacity.¹⁷ This paper has touched on the advancements that shape changed solid nanoparticles have had on biotechnology (Section 3.1), which is the first step in many therapeutic treatments.^29,62 The next step is the use of shape-modulated polymersomes to deliver drugs in a safe and effective way. Rodshaped polymersomes have been tested for their ability to sufficiently target, circulate, adhere, release, and internalize both hydrophilic and hydrophobic drugs. Work done by L’Amoreaux et al. shows that these prolate-shaped nanoparticles not only carry a wider range of drugs but also are taken up by human neural cells (SH-SY5Y) in vitro more efficiently than their spherical counterparts.²⁰ By the use of hydrophobic model molecules, results are promising that other hydrophobic anticancer drugs like paclitaxel and doxorubicin can be loaded into these multifaceted vessels and used as a minimally invasive treatment.

4. CONCLUSIONS AND OUTLOOK

This paper highlights the major benefits of modulating nanoparticle shape to enhance uptake, mainly focusing on the benefits of the elongation of nanoparticles. Although elongated solid nanoparticles have many advantages, missing is the ability to encapsulate amphiphilic payloads and add targeting ligands for uptake specificity without decreasing the therapeutic payload. By merging shape modulation techniques with the benefits of amphiphilic, stimuli-responsive polymersomes, the field would gain the benefits of both, leading to more targeted, specific, and personalized medicine. The combination of stimuli-responsive behavior with elongation can cause pathology-based drug delivery with higher probabilities of cellular uptake. Although some methods for modulating the shape of polymersomes have been explored, there seems to be no standardized protocols.

The advent of polymersome shape modulation methods presented here and a strong thermodynamic understanding of polymersome shape modulation calls for protocols to develop target-specific polymersome technologies that could have a massive impact in the treatment of disease. The techniques used can vary in success based on the hydrophobicity of the block copolymer used to create the polymersome, necessitating alternative strategies for each therapeutic application involving different polymeric materials. In the field of drug delivery, stimuli-responsive polymersomes have the benefit of responding to pathology, and this benefit should be maintained when elongating the polymersome shape. As drug delivery advances in this direction, clear protocols for the shape modulation of polymersomes need to be established.

Another important focus for the field should be the study of the effect of shape modulation on drug payloads, targeting, and thermodynamic nanoparticle stability. Decreased inner volumes, although leading to beneficial cellular uptake behavior, could lead to decreased drug payloads. Therefore, shape-modulated polymersomes need to be optimized for the end application, ensuring maintenance of drug concentrations in the therapeutic window for each given drug product. The addition of surface targeting ligands should multiply the benefit of elongation (Figure 7), especially when polymersome–cell interactions occur on the elongated edge (R₂). Our group has looked at stability of shape-modulated polymersomes in isotonic solution over a period of 7 days,²⁰ but more extensive studies on the long-term stability of these altered polymersomes need to be performed before in vivo application. To this point, modulation of polymersome shape has mainly been focused on the creation of pseudocells, with a missing focus on the potential implications on the drug delivery field.

Figure 7.

Targeting ligands attached to an elongated polymersome, showing the increased surface area capable of interacting with the cell membrane.